Biomedical subjects

A Bernstein

Publications and source records attributed to A Bernstein.

At least 19 recordsLinked to original sources

PubMed1995

Each domain of the N-ethylmaleimide-sensitive fusion protein contributes to its transport activity.

N-Ethylmaleimide-sensitive fusion protein (NSF) has been shown to be involved in numerous intracellular transport events. In an effort to understand the basic mechanism of NSF in vesicle-target membrane fusion events, we have examined the role that each of its three domains play in how NSF interacts with the SNAP.SNARE complex. Mutagenesis of the first ATP-binding domain (D1, amino acids 206-477) demonstrates that nucleotide binding by this domain is required for 20 S particle assembly. A second mutation, which permits ATP binding but not hydrolysis, yields a protein that can form 20 S particle but fails to mediate its disassembly. Similar mutations of the second ATP-binding domain (D2, amino acids 478-744) result in trimeric molecules that behave like wild type NSF. Domain rearrangement mutants were used to further probe the functional role of each domain. The amino-terminal domain (N, amino acids 1-205) is absolutely required for binding of NSF to the SNAP.SNARE complex, because the truncated mutant, D1D2, is unable to form 20 S particle. When tested as an isolated recombinant protein, the N domain is not sufficient for binding to the SNAP.SNARE complex, but when adjacent to the D1 domain or in a trimeric molecule, the N domain does mediate binding to the SNAP.SNARE complex. Monomeric N-D1 and trimeric N-D2 could both participate in particle formation. Only the N-D1 mutant was able to facilitate MgATP-dependent release from the SNAP.SNARE complex. These data demonstrate that NSF binding to the SNAP.SNARE complex is mediated by the N domain and that both ATP binding and hydrolysis by the D1 domain are essential for 20 S particle dynamics. The intramolecular interactions outlined suggest a mechanism by which NSF may use ATP hydrolysis to facilitate the vesicle fusion process.

Adenosine Triphosphate↗

PubMed1995

Defective T-cell receptor signalling and positive selection of Vav-deficient CD4+ CD8+ thymocytes.

During lymphocyte development, cellular proliferation and positive and negative selection events ensure the production of T and B lymphocytes bearing highly diverse, but self-tolerant, repertoires of antigen receptors. These processes are initiated when engagement of growth-factor receptors, or the T and B lymphocyte antigen receptors, induces tyrosine phosphorylation of specific SH2- and SH3-domain-containing cytoplasmic proteins, including Vav. Here we show that vav-/- embryonic stem cells generate only limited numbers of immature and mature T and B lymphocytes in the RAG-2 blastocyst complementation assay. Furthermore, Vav-deficient T lymphocytes showed severely impaired antigen receptor signalling. Finally, we demonstrate that Vav-dependent signalling pathways regulate maturation, but not CD4/CD8 lineage commitment, during T-cell-receptor-mediated positive selection of immature CD4+ CD8+ precursors into mature CD4+ CD8- or CD4- CD8+ T cells.

Animals↗

PubMed1995

W/kit gene required for interstitial cells of Cajal and for intestinal pacemaker activity.

The pacemaker activity in the mammalian gut is responsible for generating anally propagating phasic contractions. The cellular basis for this intrinsic activity is unknown. The smooth muscle cells of the external muscle layers and the innervated cellular network of interstitial cells of Cajal, which is closely associated with the external muscle layers of the mammalian gut, have both been proposed to stimulate pacemaker activity. The interstitial cells of Cajal were identified in the last century but their developmental origin and function have remained unclear. Here we show that the interstitial cells of Cajal express the Kit receptor tyrosine kinase. Furthermore, mice with mutations in the dominant white spotting (W) locus, which have cellular defects in haematopoiesis, melanogenesis and gametogenesis as a result of mutations in the Kit gene, also lack the network of interstitial cells of Cajal associated with Auerbach's nerve plexus and intestinal pacemaker activity.

Action Potentials↗

PubMed1995

A 1.8-Mb YAC contig spanning three members of the receptor tyrosine kinase gene family (Pdgfra, Kit, and Flk1) on mouse chromosome 5.

We constructed a yeast artificial chromosome (YAC) contig spanning the genes encoding Kit (Kit), the platelet-derived growth factor alpha receptor (Pdgfra), and fetal liver kinase 1 (Flk1), three members of a receptor tyrosine kinase gene family located in the central portion of mouse chromosome 5. The orientation of YAC clones and the extent of their overlap was determined by "probe content mapping," that is, hybridization analysis of YAC clones using the available gene probes and YAC end sequences. For four YAC clones, which constitute a minimal set spanning 1.8 Mb, a detailed restriction map was constructed. This map, in conjunction with the previously published long-range restriction map, indicates the order, the physical distances, and the relative transcriptional orientations of the Pdgfra, Kit, and Flk1 genes. The YAC clones and corresponding YAC end probes presented here provide an important resource for the molecular analysis of a cluster of developmental mutations, namely dominant white spotting (W), patch (Ph), recessive spotting (rs), and rump-white (Rw), associated with this chromosomal region.

Animals↗

PubMed1995

The vav proto-oncogene is required early in embryogenesis but not for hematopoietic development in vitro.

Previous studies have suggested that the vav protooncogene plays an important role in hematopoiesis. To study this further, we have ablated the vav protooncogene by homologous recombination in embryonic stem (ES) cells. Homozygous vav (-/-) ES clones differentiate normally in culture and generate cells of erythroid, myeloid and mast cell lineages. Mice heterozygous for the targeted vav allele do not display any obvious abnormalities. However, homozygous embryos die very early during development. Crosses of vav (+/-) heterozygous mice yield apparently normal vav (-/-) E3.5 embryos but not post-implantation embryos (> or = E7.5). Furthermore, homozygous vav (-/-) blastocysts do not hatch in vitro. These results indicate that vav is essential for an early developmental step(s) that precedes the onset of hematopoiesis. Consistent with the phenotypic analysis of vav (-/-) embryos, we have identified Vav immunoreactivity in the extra-embryonic trophoblastic cell layer but not in the inner embryonic cell mass of E3.5 preimplantation embryos or in the egg cylinder of E6.5 and E7.5 post-implantation embryos. These results suggest that the vav gene is essential for normal trophoblast development and for implantation of the developing embryo.

Animals↗

PubMed1995

Apoptosis, cancer and the p53 tumour suppressor gene.

One of the most commonly detected abnormalities in human cancer is mutation of the p53 tumour suppressor gene. Intrinsic to the function of p53 is its ability to induce apoptotic cell death and to cause cell cycle arrest. Moreover, p53 plays an important role in controlling the cellular response to DNA damaging agents such as ionizing radiation and cancer chemotherapeutic drugs. Loss of p53 function causes increased resistance to radiation and chemotherapeutic agents, and there is increasing evidence that p53 mutational status is an important determinant of clinical outcome in cancer. This review will focus on recent data describing the biochemistry of p53 function, its role in mediating apoptosis and cell cycle arrest and in the control of tumour growth and death.

Animals↗

PubMed1995

Expression of Xkl-1, a Xenopus gene related to mammalian c-kit, in dorsal embryonic tissue.

In mice, the Kit receptor tyrosine kinase and its ligand, Steel factor, are required for melanogenesis, hematopoesis and gametogenesis We have identified a Xenopus gene, Xkl-1 (Xenopus Kit-like-1) whose predicted protein has striking sequence identity in the catalytic domain and kinase insert to that of c-kit. Xkl-1 is expressed only in dorsal tissues such as the nervous system, notochord and somites of neurulae. Ultraviolet irradiated embryos and animal caps treated with basic FGF unexpectedly express Xkl-1, since they are considered to develop only ventral type tissues. These observations raise the hypothesis that Xkl-1 is involved in Xenopus dorsal development and that dorsal tissues inhibit the expression of Xkl-1 in ventral structures.

Amino Acid Sequence↗

PubMed1995

A comparative study of the application of scanning acoustic microscopy and confocal laser scanning microscopy to the structural assessment of human bones.

The Scanning Acoustic Microscope (SAM) is used to image mechanical structure and to measure the sound velocity in micrometers of in vitro samples from human bone. A similar principle is used in confocal laser scanning microscopy. The same sample is imaged by both methods and the results compared. We concentrated on the SAM, the function of which is described in greater detail. We demonstrate that the confocal scanning principle is applied in both microscopes, although information about structure differs, depending on the method used.

Acoustics↗

PubMed1995

A mutant p53 transgene accelerates tumour development in heterozygous but not nullizygous p53-deficient mice.

To test the behaviour of a mutant form of p53 in the presence and absence of wild-type p53 in vivo, we mated p53-deficient mice containing a p53 null allele to transgenic mice containing multiple copies of a mutant p53 gene (Val 135). Animals hemizygous for the endogenous wild-type p53 gene with the mutant transgene exhibited accelerated tumour development and an altered tumour spectrum compared to their non-transgenic counterparts. In contrast, transgenic and non-transgenic animals nullizygous for endogenous p53 developed tumours at the same rate. Thus, the mutant Val-135 p53 allele may act in vivo in a dominant negative manner in the presence of wild-type p53 but does not display gain of function activity in the absence of wild-type p53.

Animals↗

PubMed1995

Dysphagia due to secondary achalasia as an early manifestation of squamous cell carcinoma.

A 59-year-old man, a smoker, presented with features of airflow obstruction due to squamous cell carcinoma of central airways mimicking chronic obstructive airways disease. He also had pronounced dysphagia. Computed tomographic and magnetic resonance imaging scans showed mediastinal tumour invasion but no direct oesophageal involvement. Oesophageal manometry studies revealed that dysphagia was due to the oesophageal motility disorder, secondary achalasia.

Bronchial Neoplasms↗

PubMed1994

DNA damage, oncogenesis and the p53 tumour-suppressor gene.

The p53 tumour-suppressor gene encodes a transcription factor which plays a central role in controlling oncogenic development in mouse and humans. Mice which over-express mutant p53 transgenes or have a homozygous deletion of the p53 gene show a high frequency of spontaneous tumour development. This review will focus on recent developments using these transgenic and null mice which suggest that p53 is important in maintaining genomic stability, and is a critical component in the cellular response to ionizing radiation.

Animals↗

PubMed1994

Expression of the Kit and KitA receptor isoforms in human acute myelogenous leukemia.

Genetic and biologic evidence suggests that the Kit receptor tyrosine kinase is important in early events in hematopoietic stem cell differentiation. Two naturally occurring isoforms of the Kit receptor, termed Kit and KitA, were originally described in mouse cells and, subsequently, in human cells. These isoforms differ by the presence (KitA) or absence (Kit) of four amino acids (Gly-Asn-Asn-Lys) that lie immediately outside the transmembrane domain. RNase protection was used to measure the levels of Kit and KitA mRNA in normal bone marrow and the blast cells from individuals with acute myelogenous leukemia (AML). Although both isoforms were present in all the AML samples tested, there was considerable heterogeneity in the relative levels of the two transcripts, with Kit to KitA RNA ratios varying from as low as 1.3 to as high as 12. In contrast, the ratio of Kit to KitA transcripts in normal bone marrow was tightly clustered between 4.4 and 5.5. Because alterations in the relative levels of expression of Kit and KitA may affect the ability of a cell to respond to the Kit ligand, Steel factor, we examined the Kit/KitA RNA ratio in AML patients that differed with respect to a number of diagnostic, prognostic, and biologic parameters. The relative levels of Kit to KitA RNA was independent of French-American-British subtype, response to therapy, and primary and secondary plating efficiencies in vitro. Thus, these data suggest that the relative levels of the two isoforms of the Kit receptor in AML are not associated with any obvious biologic or clinical parameters and, therefore, may reflect naturally occurring changes in splicing mechanisms as stem cells differentiate.

Amino Acid Sequence↗

PubMed1994

Forearm compartment syndrome following brachial arterial puncture in uremia.

Although compartment syndromes of the forearm are infrequently encountered, they are well described in the literature. Forearm compartment syndrome uncommonly occurs after percutaneous arterial blood sampling and is usually associated with anticoagulant therapy. Our review of the English literature revealed no other cases of forearm compartment syndrome after arterial blood sampling in patients with bleeding diathesis due to chronic renal failure. This article discusses a 30-year-old woman with Good-pasture's syndrome who developed a compartment syndrome after a brachial artery blood gas. The cause, diagnosis, and treatment of compartment syndrome and uremic bleeding, as well as a review of the literature, are discussed.

Adult↗

PubMed1994

The Fps/Fes protein-tyrosine kinase promotes angiogenesis in transgenic mice.

The fps/fes proto-oncogene encodes a cytoplasmic protein-tyrosine kinase known to be highly expressed in hematopoietic cells. To investigate fps/fes biological function, an activating mutation was introduced into the human fps/fes gene which directs amino-terminal myristylation of the Fps/Fes protein. This mutant, myristylated protein induced transformation of Rat-2 fibroblasts. The mutant fps/fes allele was incorporated into the mouse germ line and was found to be appropriately expressed in transgenic mice, in a tissue-specific pattern indistinguishable from that of the endogenous mouse gene. These mice displayed widespread hypervascularity, progressing to multifocal hemangiomas. High levels of both the transgenic human and endogenous murine fps/fes transcripts were detected in vascular tumors by using RNase protection, and fps/fes transcripts were localized to endothelial cells of both the vascular tumors and normal blood vessels by in situ RNA hybridization. Primary human umbilical vein endothelial cultures were also shown to express fps/fes transcripts and the Fps/Fes tyrosine kinase. These results indicate that fps/fes expression is intrinsic to cells of the vascular endothelial lineage and suggest a direct role of the Fps/Fes protein-tyrosine kinase in the regulation of angiogenesis.

Amino Acid Sequence↗

PubMed1994

Assessing drug use prevalence in the workplace: a comparison of self-report methods and urinalysis.

A random sample of 800 employees of a steel manufacturing company were randomly assigned to one of four self-report methods of assessing illicit drug use: 1) Individual interview in the workplace, 2) group-administered questionnaire in the workplace, 3) telephone interview, and 4) individual interview off the worksite. All 621 subjects participating in the research were also tested by urinalysis. Rates of drug use self-report were highest in the workplace interview condition and lowest in the overall group questionnaire condition. Although the overall prevalence rates produced by self-reports and urinalysis were similar, there was little concordance between urinalysis positives and self-report positives. The results indicated that self-reports and urinalysis are complementary methods of drug use assessment, and are best used in combination.

Employment↗

PubMed1994

Susceptibility to radiation-carcinogenesis and accumulation of chromosomal breakage in p53 deficient mice.

The p53 tumour suppressor gene is an important participant in the cellular response to ionizing radiation and other DNA damaging agents. Cells which lack p53 are unable to arrest cell cycle or enter into apoptotic cell death following irradiation. Moreover, these p53 deficient cells exhibit an increased resistance to DNA damaging agents, including radiation. The significance of this radiation-resistance and its relationship to the role that p53 plays in tumour suppression and the cellular radiation response has not yet been determined. In this report we have analyzed p53 deficient mice, expressing either a mutant p53 transgene or having a targeted p53 null allele, in order to investigate the role that p53 plays in governing susceptibility to radiation-carcinogenesis and in controlling the in vivo accumulation of chromosomal abnormalities. We show that wild-type p53 plays a critical role in controlling susceptibility to gamma-radiation-induced tumorigenesis, and sarcomas and lymphomas rapidly appear in irradiated p53 transgenic mice. Moreover, this susceptibility to radiation-carcinogenesis is associated with a two-fold increase in the in vivo accumulation of radiation-induced double stranded chromosomal breaks relative to that observed in wild-type animal. Taken together, these observations suggest that p53 acts to suppress tumour formation in vivo by preventing the accumulation of cells that have sustained radiation-induced DNA damage.

Animals↗

PubMed1993

Dynamic changes in ovarian c-kit and Steel expression during the estrous reproductive cycle.

W and Steel mutant mice exhibit similar developmental defects in melanogenesis, haematopoiesis, and gametogenesis. Consistent with the cell autonomous and microenvironmental nature of W and Sl mutations, respectively, W encodes the c-kit receptor tyrosine kinase while Steel encodes the Kit ligand. Both c-kit and Steel are expressed in various cells in which no corresponding mutant phenotype has yet been demonstrated. In the adult ovary, certain stromal-derived cells (theca and interstitial), as well as oocytes, express c-kit, while granulosa cells express Steel. We show here that the cessation of oocyte growth, at the transition of the follicle to the antral stage, is associated with the cessation of Steel expression in the cumulus granulosa cells in the vicinity of the oocyte. These observations suggest a role for the Kit signaling pathway in oocyte growth or in meiotic arrest. In addition, the cyclic secretion of luteinizing hormone immediately and dramatically results in elevated Steel expression in mural granulosa cells and decreased levels of c-kit transcripts in stromal-derived cells. This influence of the estrous reproductive cycle on c-kit/Steel expression suggests that the Kit signaling pathway, in addition to its previously described role in primordial germ cell development, is involved in follicular development in the adult female.

Animals↗

PubMed1993

Molecular genetic approaches to the elucidation of hematopoietic stem cell function.

The past few years have seen considerable advances in the development of the methodologies for discovering novel genes critical to hematopoietic stem cell function and for analyzing their biological role in hematopoiesis. This review briefly discusses some common themes that are emerging from the molecular genetic approaches to hematopoietic stem cell function.

Anemia↗